Mechanism of YopJ-Induced Cell Death and Capase-1 Activation in Macrophages Infected with Yersinia

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Pathogenic Yersinia species encode a Type III Secretion System (T3SS) that translocates a group of effectors termed as Yersinia outer proteins (Yops) into target cells to disrupt eukaryotic host signaling pathways. YopJ is an acetyltransferase, which prevents MAP Kinase Kinase (MKK) and I?ÂB Kinase ? (IKK? ) phosphorylation and activation and causes Toll like receptor 4 (TLR4)-directed apoptosis in macrophages. YopJ isoforms encoded by different Yersinia strains exhibit a range of cytotoxic activities. In previous work, a YopJ isoform in Y. pestis strain KIM5 (YopJKIM) was shown to cause enhanced cytotoxicity, caspase-1 activation and interleukin-1? (IL-1? ) release in infected macrophages. In my study, the molecular basis for elevated activity of YopJKIM was determined. In addition, the mechanism of cell death and caspase-1 activation in KIM5-infected macrophages was examined. Finally, I investigated the role of caspase-1 in a protective host response to Yersinia strains with normal or enhanced cytotoxicity in mice. I determined using site directed mutagenesis that two amino acid substitutions, F177L and K206E, in YopJKIM are important for enhanced apoptosis, caspase-1 activation, and IL-1? release in Yersinia-infected macrophages. Despite secretion and translocation levels that were similar to other YopJ isoforms (e.g. YopJCO92 (F177, K206) or YopJYPTB (F177)), YopJKIM displayed an up-regulated capacity to inhibit phosphorylation of NF-?ÂB inhibitor ? (I?ÂB? ) and a moderately raised ability to inhibit phosphorylation of mitogen-activated protein kinase (MAPK). YopJKIM also had enhanced binding to substrate IKK? as compared to YopJCO92 by pull down assay. Previous work had established a role for IKK? in the negative regulation of caspase-1 activation in LPS-stimulated macrophages. I demonstrated that IKK? negatively regulates caspase-1 activation in response to a live pathogen by infection of Ikk? ? or wild type macrophages with KIM5. Significantly increased caspase-1 activation was identified in Ikk? ? cells, as compared to wild type macrophages by FLICA staining. A small molecule IKK? inhibitor, TPCA-1, was adopted to mimic YopJ function. Following TLR4 activation with LPS, cell death and IL-1? release occurred in macrophages treated with TPCA-1, highlighting the importance of IKK? in negative regulation of caspase-1. In my studies of the mechanism of cell death in KIM5-infected macrophages, low caspase-3/-7 activity was detected by luminol assay and Poly (ADP-ribose) polymerase (PARP) cleavage. Cytotoxicity and IL-1? release were not reduced by caspase-8 inhibitor treatment or the use of bax/bak double knockout (DKO) macrophage infection, indicating that cell death and caspase-1 activation were independent of canonical apoptosis pathways. Alternatively, assays for High mobility group box 1 (HMGB1) release and annexin V/ propidium iodide (PI) staining suggested necrotic macrophage death during KIM5 infection. Staining for active Caspase-1/PI uptake was performed and results showed that caspase-1 activation was associated with macrophages undergoing cell death, suggesting that caspase-1 activation may be related to necrosis. Inhibitor studies showed that reactive oxygen species (ROS) and receptor interacting protein 1 (RIP1) were not required for cytotoxicity and IL-1? production. On the other hand, cathepsin B inhibitors significantly reduced IL-1? release and caspase-1 activation, implicating cathepsin B leakage from ruptured lysosomes in caspase-1 activation. Cytotoxicity was not reduced by cathepsin B inhibitors, confirming that caspase-1 is not required for cell death in KIM5-infected macrophages. Finally, different YopJ isoforms were ectopically expressed in Y. pseudotuberculosis and YopP was determined to have the highest capacity to cause cytotoxicity and activate caspase-1 in infected macrophages. Y. pseudotuberculosis strains ectopically expressing YopJYPTB or YopP were used to infect wild type or caspase-1 deficient mice to study the protective role of caspase-1. Results show that caspase-1 is dispensable for the protective innate immune response against Yersinia with normal (YopJYPTB) or enhanced (YopP) cytotoxicity. In summary, my results demonstrate that enhanced cytotoxicity and caspase-1 activation in KIM5-infected macrophages result from two amino acid replacements in YopJ, which intensifies its ability to inhibit nuclear factor ?ÂB (NF-?ÂB) and mitogen-activated protein kinase (MAPK) signaling pathways. The enhanced necrotic cell death is apparently sensed by the inflammasome leading to caspase-1 activation and IL-1? release in macrophages. However, caspase-1 is not required for a protective innate immune response to Yersinia with normal or enhanced cytotoxicity in mice.